This application is the national phase of international application PCT/GB98/02156 filed Jul. 17, 1998 which designated the U.S.
The present invention concerns immunogenic epitopes of Shigella-like toxins (SLTs), particularly the Shigella-like toxin of E. coli O157:H7, their use as immunogens and in treatment or diagnosis, agents (for example antibodies and antigen-binding fragments) which specifically neutralise them, their use in treatment and diagnosis, and methods for same.
Shigella-like toxins (also known as Shiga-like toxins and Vero toxins) are well known (Schmitt, C. K. et al, 1991, Infection and Immunity, 59 (13): 1065-1073) and are produced by a wide range of pathogens including E. coli O157:H7, infection causing bloody diarrhoea and acute kidney failure, with many patients, particularly the young and elderly, failing to survive an infection. Outbreaks are sporadic but of significant size and present a substantial burden on health care resources (Berkelman, R. L. et al., 1994, Science, 264: 368-370; Slutsker, L. et al., 1997, Ann. Intern. Med., 126: 505-513). In the US alone, an estimated 20,000 cases of E. coli O157:H7 infection occur annually. Infection frequently occurs as a result of consuming contaminated foods, particularly ground beef products such as hamburgers, and by person-to-person contact in child care centres. Other reported outbreaks have occurred in Scotland and Japan (1996, BMJ, 313: 1424), the Pacific North West (Antibiotic-Resistant Bacteria, Office of Technology Assessment, Congress of the United States, pp 150-151) and Canada (Slutsker, L. et al., 1997, Ann. Intern. Med., 126: 506-513), although outbreaks are in no way limited to these regions.
Many of the infecting pathogens have acquired multiple drug resistance, and it has been found that antibiotic treatment may cause the bacteria to increase the production of the Shigella-like toxin (Antibiotic-Resistant Bacteria, supra). A need for novel therapeutics for pathogens expressing Shigella-like toxins has been felt for many years (see for example Antibiotic-Resistant Bacteria, supra). It has been suggested (Antibiotic-Resistant Bacteria, supra) that antibodies specific against the Shigella-like toxin of E. coli O157:H7 may have therapeutic potential, but to date antibodies have not been used therapeutically.
Various Shigella-like toxins have been cloned and sequenced (Meyer, T. et al., 1922, Zbl. Bakt., 276: 176-188, Schmitt, C. K. et al., 1991, Infection and Immunity, 59 (3): 1065-1073, Ramotar, K. et al., 1995, J Clin. Microbiol, 33 (3): 519-524). However, immunogenic regions, particularly specific epitopes of the toxins have not been identified.
The present inventors have now succeeded in identifying a number of epitopes from E. coli Shigella-like toxins, particularly those of E. coli O157:H7. The epitopes have a wide range of usesxe2x80x94they may be used therapeutically as immunogens, for example as vaccines, or diagnostically to detect agents (e.g. antibodies) which bind specifically to them. They may also be used to produce neutralising agents, for example antibodies, which neutralise the toxin. Agents which bind the epitopes may be used both therapeutically and diagnostically.
According to the present invention there is provided an epitope of a Shigella-like toxin, having a sequence selected from any one of the group of SEQ ID NOs: 1-7. The epitope may have a sequence selected from either one of SEQ ID NOs: 1 and 3. The epitopes of the present invention may also be described as peptides carrying epitopes of a Shigella-like toxin.
The epitopes of SEQ ID NOs: 1-7 have not been previously identified, nor have they been suggested. Although the sequences of various SLTs are known, specific epitopes are not.
The epitopes of the present invention are also considered to encompass analogues of the epitopes. Analogues may be readily produced, for example in the form of mimotopes (Geysen, H. M. et al., 1987, Journal of Immunological Methods, 102: 259-274; Geysen, H. M. et al.,1988, J. Mol. Recognit., 1(1):32-41; Jung, G. and Beck-Sickinger, A. G., 1992, Angew. Chem. Int. Ed. Eng., 31: 367-486) using commercially available mimotope design technology.
The Shigella-like toxin may be that from an E. coli. It may be that from an E. coli O157 selected from the group of O157:H7, O157:Hxe2x88x92 and O26:H11. Alternatively, the Shigella-like toxin may be selected from the group of that of Shigella sonnei, Shigella boydii, Shigella flexneri, and Shigella dysenteriae. 
The epitope may be for use in a method of treatment or diagnosis of the human or animal body.
The epitope may for example be for use as an immunogen, for example a vaccine.
Also provided according to the present invention are binding agents specific against an epitope according to the present invention. Binding agents include any molecule which is capable of recognising an epitope according to the present invention. For example a binding agent may be an antibody or an antigen binding fragment thereof.
Antibodies are well known (Harlow, E. and Lane, D., xe2x80x9cAntibodiesxe2x80x94A Laboratory Manualxe2x80x9d, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, New York, 1988). The antibody may be a whole antibody or an antigen binding fragment thereof and may in general belong to any immunoglobulin class. Thus, for example, it may be an IgM or an IgG antibody. The antibody or fragment may be of animal, for example, mammalian origin and may be for example of murine, rat, sheep or human origin. It may be a natural antibody or a fragment thereof, or, if desired, a recombinant antibody fragment, i.e. an antibody or antibody fragment which has been produced using recombinant DNA techniques.
Particular recombinant antibodies or antibody fragments include, (1) those having an antigen binding site at least part of which is derived from a different antibody, for example those in which the hypervariable or complementarity determining regions of one antibody have been grafted into the variable framework regions of a second, different antibody (as described in, for example, EP 239400); (2) recombinant antibodies or fragments wherein non-Fv sequences have been substituted by non-Fv sequences from other, different antibodies (as described in, for example, EP 171469, 173494 and 194276); or (3) recombinant antibodies or fragments possessing substantially the structure of a natural immunoglobulin but wherein the hinge region has a different number of cysteine residues from that found in the natural immunoglobulin but wherein one or more cysteine residues in a surface pocket of the recombinant antibody or fragment is in the place of another amino acid residue present in the natural immunoglobulin (as described in, for example, PCT/GB88/00730 and PCT/GB88/00729).
The antibody or antibody fragment may be of polyclonal or monoclonal origin. It may be specific for at least one epitope.
Antigen binding antibody fragments include, for example, fragments derived by proteolytic cleavage of a whole antibody, such as F(abxe2x80x2)2, Fabxe2x80x2 or Fab fragments, or fragments obtained by recombinant DNA techniques, for example Fv fragments (as described, for example, in PCT/GB88/0747).
The antibodies according to the invention may be prepared using well-known immunological techniques. Thus, for example, any suitable host may be injected with the protein and the serum collected to yield the desired polyclonal antibody after appropriate purification and/or concentration (for example by affinity chromatography using the immobilised protein as the affinity medium). Alternatively splenocytes or lymphocytes may be recovered from the protein-injected host and immortalised using for example the method of Kohler et al. (1976, Eur. J. Immunol., 6: 511), the resulting cells being segregated to obtain a single genetic line producing monoclonal antibodies. Antibody fragments may be produced using conventional techniques, for example, by enzymatic digestion with pepsin or papain. Where it is desired to produce recombinant antibodies according to the invention these may be produced using, for example, the methods described in EP 171469, EP 173494, EP 194276 and EP 239400.
Antibodies according to the invention may be labelled with a detectable label or may be conjugated with an effector molecule, for example a drug eg. an antibacterial agent or a toxin or an enzyme, using conventional procedures and the invention extends to such labelled antibodies or antibody conjugates. Thus a binding agent according to the present invention may not only bind to the Shigella-like toxin but may also neutralise it (i.e. inhibit its cytotoxicity).
Binding agents according to the present invention may be for use in a method of treatment or diagnosis of the human or aniomal body.
Also provided according to the present invention is the use of an epitope or binding agent according to the present invention in the manufacture of a medicament for the treatment of a condition resulting from a Shigella-like toxin. Also provided is a method of manufacture of a medicament for the treatment of a condition resulting from a Shigella-like toxin, comprising the use of an epitope or binding agent according to the present invention.
Thus the present invention provides epitopes of Shigella-like toxins, which may be used therapeutically or diagnostically, together with binding agents derived therefrom which themselves may be used both diagnostically or therapeutically.
Also provided according to the present invention is a diagnostic test method for a Shigella-like toxin displaying an epitope according to the present invention comprising the steps of:
i) reacting a binding agent according to the present invention with a sample
ii) detecting a binding agent-epitope binding reaction; and
iii) correlating the detection of the binding reaction with the presence of the Shigella-like toxin.
The binding agent may for exampe be an antibody, and the diagnostic test method comprise the steps of:
i) reacting an antibody according to the present invention with a sample;
ii) detecting an antibody-antigen binding reaction; and
iii) correlating the detection of the binding reaction with the presence of the Shigella-like toxin.
Where the binding agent is an antibody or antigen binding fragment thereof, the epitopes may be regarded as being antigens.
Also provided is a diagnostic test method for antibody specific against a Shigella-like toxin comprising the steps of:
i) reacting an epitope according to the present invention with a sample;
ii) detecting an antibody-antigen binding reaction; and
iii) correlating the detection of the antibody-antigen binding reaction with the presence of antibody specific against a Shigella-like toxin.
The sample may be from a patient, for example a serum sample or a peritoneal dialysate, although any other sample which may contain, or which might be expected to contain, Shigella-like toxins may of course be used.
Also provided according to another aspect of the present invention is a diagnostic test kit for performing a diagnostic test method according to the present invention. Diagnostic test kits are well known and may for example include dip-stick tests according to WO 88/08534. The test kit may include instructions for its use in a diagnostic test method according to the present invention.
Also provided according to the present invention is a method of treatment or diagnosis of the human or animal body comprising the use of an epitope or binding agent according to the present invention.
Medicaments and methods of treatment according to the present invention will be readily apparent to one skilled in the art. Medicaments may be prepared using pharmaceutically acceptable carriers, diluents or excipients (Remington""s Pharmaceutical Sciences and US Pharmacopeia (1984) Mack Publishing Company., Easton, Pa., USA). The medicaments and methods of treatment may be effected using a pharmaceutically effective amount of the epitope or binding agent. Appropriate dosages will be readily apparent to one skilled in the art and may be readily determined, for example by means of dose-response experiments.